BDNF down-regulates neurotrophin responsiveness, TrkB protein and TrkB mRNA levels in cultured rat hippocampal neurons.

Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal Trkb protein does not appear to be just a development phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro, this leads to long-term functional desensitization to BDNF, NT-3 and NT-4/5, as well as down-regulation of trkB and trkC mRNA.

A quantitative method for morphometric analysis in neuronal cell culture: unbiased estimation of neuron area and number of branch points.

The morphology and fine structure of neurons in vivo as well as in vitro are influenced by a variety of cell-adhesion and extracellular matrix molecules and soluble growth factors. To examine the effects of such molecules, we have developed a new method for the quantitation of several parameters associated with the morphology of neurons in culture. Whereas methods which have been traditionally used to perform quantitative morphometric analysis of neurons in vitro are often time-consuming and subjective, the methods we describe provide a rapid, efficient, and unbiased approach to morphometric analysis of cultured neurons.

The neurotrophins BDNF, NT-3 and NT-4/5 promote survival and morphological and biochemical differentiation of striatal neurons in vitro.

The neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5) and nerve growth factor (NGF), were compared for their effects on the survival and differentiation of embryonic rat striatal neurons grown in low-density cultures. Treatment with BDNF for 8 days resulted in a 40% increase in overall neuronal survival, a 3- to 5-fold increase in the number of calbindin-immunoreactive neurons, and an 80% increase in GABA-positive neurons. Treatment with NT-3 or NT-4/5 produced a 2- to 3-fold increase in the number of calbindin-positive neurons and an increase in GABA-positive cell number similar to that induced by BDNF, BDNF treatment produced a striking morphological differentiation of striatal GABAergic neurons, which was characterized by a doubling of the number of neurite branch points, the total area of aborization and the perikaryal area compared to control cultures. All three of these factors increased high-affinity GABA uptake 2-fold. NGF had no effect on any of the parameters examined. Our results show that BDNF, NT-3 and NT-4/5 promote the survival and/or differentiation of calbindin-immunopositive and GABAergic striatal neurons.

Somatostatin gene regulation: an overview.

The somatostatinergic system has proven to be one of the best models of neuropeptide biology. Originally characterized as a hypothalamic regulator of growth hormone secretion, somatostatin also regulates the secretion of several other pituitary, pancreatic, and gastrointestinal (GI) hormones including thyrotropin-stimulating hormone, insulin, glucagon, and gastrin. Disorders in somatostatin metabolism have been proposed to contribute to the pathogenesis of Alzheimer's disease, epilepsy, GI motility disorders, and diabetes. On a more basic level, studies of somatostatin action have integrated divergent concepts of intracellular signal transduction. Advances in the understanding of somatostatin biosynthesis have had an impact on areas outside the field of endocrinology by providing new concepts of eukaryotic gene regulation. This report focuses on the transcriptional regulation of somatostatin gene expression. Two aspects of somatostatin gene transcription will be considered--regulated expression by second messengers and tissue-specific basal expression.

Processing and intracellular sorting of anglerfish and rat preprosomatostatins in mammalian endocrine cells.

Rat preprosomatostatin (rPPSS) is processed to two distinct end products in a tissue-specific manner. The analogous end products in anglerfish are derived from separate precursors, anglerfish preprosomatostatins-1 and -2 (a(1)PPSS and a(II)PPSS). This report reviews experiments demonstrating that in mammalian cells, the cell of expression, not precursor structure, determines the processing fate of the preprosomatostatins. A fusion precursor of a(II)PPSS and rPPSS was expressed in mammalian cell lines to determine that the amino-terminal 78 residues of rPPSS contain a sorting signal that directs the precursor into a regulated secretory pathway wherein proteolytic processing occurs. Preliminary studies of rPPSS pro-region mutations are presented that attempt to further localize this sorting signal.

Cultured rat neurons and astrocytes express immunologically related epitopes of the GABAA/benzodiazepine receptor.

gamma-Aminobutyric acid (GABA) is the major inhibitory transmitter in the mammalian central nervous system (CNS) and an understanding of the development of receptors for this compound is of considerable interest. In this study, 3 monoclonal antibodies directed against the GABAA/benzodiazepine (GABA/BDZ) receptor were utilized in immunocytochemical experiments to localize receptor sites on neurons in dissociated cultures from fetal rat hypothalamus. Immunoreactivity was restricted to distinct patches on the plasma membrane of neurons. Analogous patches were observed on the surface of type 1 astrocytes, but not on type 2 astrocytes or oligodendrocytes. In membrane extracts from whole brain these antibodies reacted with two proteins (50 and 55 kDa) whereas extracts of cultured astrocytes yielded a single protein of approximately 63 kDa. Thus the identity of the glial protein remains unclear, but this study provides evidence for the presence of shared epitopes of the GABA/BDZ-receptor on both neurons and glia, and suggests that the astrocytic receptors may be important to nervous system function. Moreover it indicates that in vitro GABA/BDZ-receptors may form aggregates which would account for the restricted patterns of GABA sensitivity reported in some electrophysiological studies.

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity.

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.

Cell types and cell-substrate interactions in serum-free dissociated cultures of rat hypothalamus.

We have developed a system for the long-term cultivation of hypothalamic neurons at low density in defined serum-free medium, and have identified the major classes of cells in these cultures. Cells prepared from the dissociation of embryonic rat hypothalami are plated, in serum-free medium, onto monolayers of cortical astrocytes. Neurons adhere and begin to extend neurites soon after plating, and survive for up to several weeks in culture. Cell-type-specific immunological markers were utilized to identify neurons and the major classes of glial cells in these cultures. The culture of hypothalamic neurons in serum-free conditions provides a valuable system for the study of the cellular basis of hypothalamic neuronal heterogeneity and functional diversity.

Localization of beta-adrenergic receptors on differentiated cells of the central nervous system in culture.

Hypothalamic neurons and cortical protoplasmic (type 1) astrocytes in dissociated cultures of rat brain express binding sites for antibodies specific for epitopes related to the beta-adrenergic receptor. Undifferentiated glial progenitor cells of the rat optic nerve do not detectably bind these antibodies, but both of the progeny [oligodendroglia and process-bearing (type 2) astrocytes] express immunologically identified beta-adrenergic receptors. Levels of receptor expression are variable: not all cells of each type express receptors nor is expression uniform on a given cell. The data suggest that cells of the central nervous system express beta-adrenergic receptors but that levels of expression may be determined by the differentiated state of the cells.